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Effect of LED Lights on Secondary Metabolites and Antioxidant

Activities in Red Pakchoi Baby Leaves
Leonel Tarcisio da Cristina Bungala,◆ Sang Un Park,◆ Bao Van Nguyen, Jinsu Lim, Kihyun Kim,
Jae Kwang Kim, Chang Ha Park, Anh Tuan Le, Yong Suk Chung,* and Hyeon Ji Yeo*
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ABSTRACT: Pakchoi (Brassica rapa subsp. chinensis) is one of the

most widely consumed vegetables in Asian countries, and it is high in
secondary metabolites. The availability, quantity, and quality of light
play a critical role in the growth and development of plants. In this
Downloaded via 218.158.71.208 on June 17, 2024 at 08:19:44 (UTC).

study, we investigated the effect of LEDs (light-emitting diodes; white,

blue, red, and red + blue) on anthocyanin, glucosinolates, and phenolic
levels in red pakchoi baby leaves. On the 24th day after sowing (DAS),
red baby pakchoi leaves were harvested, and shoot length, root length,
and fresh weight were measured. Among the different LED treatments,
there was no significant difference in shoot length, whereas the highest
root length was achieved in the red + blue LED treatment (23.8 cm).
The fresh weight also showed a significant difference among the
different LED treatments. In total, 12 phenolic and 7 glucosinolate
individual compounds were identified using high-performance liquid chromatography (HPLC) analysis. The highest total
glucosinolate (2937 μg/g dry wt) and phenolic (1589 μg/g dry wt) contents were achieved in baby leaves exposed to red + blue
light. Similarly, the highest contents of total anthocyanins (1726 μg/g dry wt), flavonoids (4920 μg/g dry wt), and phenolics (5900
μg/g dry wt) were achieved in the red + blue treatment. Plants exposed to red + blue LED light showed the highest accumulation of
anthocyanin, glucosinolates, and phenolic compounds. For antioxidant activity, DPPH (2,2-diphenyl-1-picrylhydrazylradical) free
radical scavenging, ABTS (2,2-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid) radical scavenging, and reducing power assays were
performed, and the antioxidant activity of red pakchoi baby leaves grown under red + blue LED light was found to be the best. The
metabolic profiling of the identified metabolites revealed distinct separation based on the secondary metabolites. This research will
be helpful for farmers to choose the best LED light combination to increase the secondary metabolic content in pakchoi plants.

1. INTRODUCTION compounds.7 The phenolic content of Brassica vegetables

Pakchoi, also known as Chinese cabbage (Brassica rapa subsp.
chinensis), belongs to the Brassicaceae family. Because of its
beneficial phytochemical composition, it is grown and
consumed annually in most Asian countries.1 It is grown and
consumed throughout the year because it has numerous
health-promoting compounds, such as anthocyanins, carote-
noids, chlorophylls, flavonoids, hydroxycinnamic acid deriva-
tives, isorhamnetin, kaempferol, phenolics, quercetin, vitamins,
and minerals.2,3
Glucosinolates are a large group of plant secondary
metabolites with nutritional effects and biological activity.
Glucosinolates are primarily found in cruciferous plants,
including the widely consumed Brassicaceae family.4 One of
the reasons for the high consumption of cruciferous vegetables
is that the breakdown products of glucosinolates, such as
nitriles, oxazolidines, thiocyanates, isothiocyanates, and epi-
thionitriles, are effective in reducing the risk of cancer and
heart failure in humans.5,6 One of the most important groups
of phytochemicals with antioxidant capacity is phenolic
© 2024 The Authors. Published by
American Chemical Society
23420
has recently been examined. Despite variations among species
and even harvests from the same species, these crops are
usually rich in phenolic compound composition.8
Among the various environmental factors, light quality is
crucial for photosynthesis, plant growth, and development.9 In
plants, light-emitting diode (LED) affects metabolites in a
significant way and plays an important role in their
physiology.10 Furthermore, LED lights are more effective
than fluorescent lights for nonheading pakchoi reproductive
and vegetative growth. Blue LED light promotes vegetative
growth, whereas red LED light promotes reproductive
growth.11 Several studies have reported that different LEDs
Received: December 28, 2023
Revised: April 30, 2024
Accepted: May 9, 2024
Published: May 22, 2024
https://doi.org/10.1021/acsomega.3c10261
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ACS Omega http://pubs.acs.org/journal/acsodf
have a significant effect on growth and secondary metabolite
production (anthocyanins, carotenoids, glucosinolates, and
phenolics) in various leafy vegetable microgreens, and sprouts
were studied such as canola sprout,6 kale,10 kohlrabi sprout,12
lettuce,13 mustard sprout,11 and Tartary buckwheat sprout.14
Baby leaf vegetables are grown in high densities, harvested,
and used when the leaves are young (juvenile stage).15 The
baby leaf market is growing rapidly owing to its convenience
and health benefits.16 The method of growing fresh vegetables
with LEDs has shown many positive aspects, such as compact
bulb material, long life, specific wavelength, and ease of
adjustment.17 In addition, LED lights have produced
antioxidant properties in various crops, such as pea seedlings,
spinach, lettuce, and komatsuna.18 According to Yeo et al.,
LED arrays increase the accumulation of secondary metabo-
lites including carotenoids, flavonoids, glucosinolates, and
phenolic acids.19
It is easier and more effective to control and evaluate the
effects of light intensity on the biosynthesis of the desired
substances.17 However, in red pakchoi baby leaves, analyses of
secondary metabolites after exposure to LED light have not
been studied well. Hence, this study aims to provide the
optimized light conditions for the plant growth, secondary
metabolite production, and antioxidant potentials of pakchoi
baby leaves via HPLC analysis of phenolics and glucosinolates
and DPPH, ABTS, and reducing power assays.
2. MATERIALS AND METHODS
2.1. Plant Materials. Red pakchoi (B. rapa subsp.
chinensis) seeds were obtained from Asia Seed Co., South
Korea. 1.0 g of seed was sterilized with 70% ethanol (v/v),
sown in an 11 cm × 11 cm plastic pot with vermiculite soil,
and exposed to white (449−551 nm), blue (452 nm), red (636
nm), and red + blue (457 and 636 nm) LED lights with a flux
rate of 90 μmol·m−2·s−1. The experiment was arranged in a
completely randomized design (CRD) with ten replications. In
an LED growth chamber (Multi-Room Chamber HB-302S-4,
Hanbaek Scientific Co., 136 cm × 78 cm × 168 cm), the
growth conditions were set at 25 °C with an 8 h dark and 16 h
light cycle. The red baby pakchoi leaves were collected 24 days
after sowing (DAS). For growth measurements, 24 DAS, ten
plants from each treatment were selected randomly, and
growth measurements were taken. The shoot length (SL) and
root length (RL) were measured in centimeters using a meter
ruler. To determine the fresh weight (FW), the pakchoi roots
were weighed in milligrams using a balance. For HPLC
examination, the samples were collected, freeze-dried at −80
°C for 72 h, and then crushed into a fine powder using a
mortar and pestle.
2.2. Glucosinolate Extraction and HPLC Analysis. 0.1
mg sample was collected in a 5 mL Eppendorf tube for
glucosinolate extraction, and 1.5 mL of 70% (v/v) methanol
was added and stirred at 70 °C. Following that, the mixers
were placed at 70 °C in a water bath for almost 5 min to
inactivate the myrosinase enzyme, after which the sample was
centrifuged for 10 min at 12,000 rpm. To obtain 4.5 mL of the
supernatant, this procedure was repeated three times. A mini-
column containing DEAE-Sephadex A-25 was loaded with the
collected supernatant, followed by the addition of 2 mL of
water with the purpose of blocking the end of the mini-column
and then adding 75 μL of arylsulfatase enzyme solution for
desulfation. Then, the mini-columns were left at room
temperature for 16 h. For HPLC analyses, desulfoglucosino-
23421
Article
lates were eluted with 0.5 mL of HPLC-grade water.
Subsequently, the prepared solutions were filtered through a
0.45 μm syringe filter. HPLC analysis and conditions were like
those in a previous study. 20 The identification and
quantification of seven glucosinolates were performed based
on our database comparing retention times, and 0.1 mg/mL
sinigrin was used as an external standard (Table S1).
2.3. Phenylpropanoid Extraction and HPLC Analysis.
A volume of 2 mL of 80% (v/v) methanol was added to 100
mg of pakchoi tissue powder samples. Subsequently, the
mixture was vortexed and then placed in a sonicator at 36 °C
for 1 h. After sonication, the samples were centrifuged at
12,000 rpm for 10 min at 4 °C to collect the supernatant.
Using 0.45 μm Whatman No. 42 filter paper, the supernatant
samples were transferred to new tubes and stored in vials for
HPLC analysis. HPLC analysis and conditions were like those
in a previous study.20 The retention times of the standard and
spike tests were used to specify the peaks in the extract. Also,
quantification was performed through each calibration curve of
12 compounds (Table S2). The standard compounds for
phenolics analysis are gallic acid (≥99%), caffeic acid (≥98%),
4-hydroxybenzoic acid (=99%), (−)-epicatechin (≥98%), rutin
(≥94%), trans-cinnamic acid (≥95%), chlorogenic acid
(≥95%), ferulic acid (=99%), catechin hydrate (≥98%), p-
coumaric acid (≥98%), quercetin (≥95%), and kaempferol
(≥97%), which were purchased from Sigma-Aldrich Co., Ltd.
(St. Louis, MO).
2.4. Measurement of Total Anthocyanin Content by
Spectrophotometry. To determine the total anthocyanin
content (TAC), 100 mg of a dry weight sample was collected
in a 5 mL tube, and then, 2 mL of 70% ethanol was added and
sonicated for 1 h. Then, the sample was centrifuged at 12,000
rpm for 20 min at 4 °C, followed by filtration into an
Eppendorf tube through a 0.45 μm PTFE hydrophilic syringe
filter. The TAC was quantified using a pH differential method
with a two-buffer system: potassium chloride buffer, pH 1.0
(25 mM), and sodium acetate buffer, pH 4.5 (400 mM).21 To
determine the total anthocyanin content, 0.4 mL of the
extraction solution was mixed with 3.6 mL of each buffer. The
absorbance of each solution was measured using the UV−vis
spectrophotometer SPECTRO star nano-plate reader (BMG
LABTECH) at 510 and 700 nm against a blank in a cuvette
with a 1 cm path length. The total anthocyanin content was
calculated as previously described. The final anthocyanin
content was expressed as cyanidin-3-glucoside equivalent
(CYE) in 1 g of dried powder (mg CYE/1 g dried powder).
For each sample extraction, the anthocyanin content was
determined in triplicate.
2.5. Total Phenolic and Total Flavonoid Content (TPC
and TFC). 100 mg of the dried sample powder was mixed with
2 mL of methanol (70%, v/v), followed by sonication for 60
min at 25 °C. The mixture was centrifuged for 20 min at
12,000 rpm and 4 °C, and then, the supernatant was filtered
through a 0.45 μm PTFE hydrophilic syringe filter. The TPC
and TFC were measured using the protocol described by
Kumla et al. and Park et al.22,23 Sample extracts were diluted
with methanol (70%, v/v) to a concentration of 5000 ppm. For
the TPC analysis, 0.5 mL of 2 N Folin and Ciocalteu phenol
reagent (Sigma-Aldrich Co., Yongin, Korea) was mixed with
0.1 mL of each extract and incubated at room temperature for
3 min. Afterward, 4 mL of 20% (v/v) Na2CO3 solution was
added to the mixture and then incubated in the dark for 90
min. The absorbances of each sample were measured at 760
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nm using the SPECTRO star Nano-plate reader (BMG and VIP (variable importance in projection) were performed
LABTECH). The total phenolic contents were quantified by using MetaboAnalyst 5.0 (http://www.metaboanalyst.ca/,
using the equivalent calibration curve of gallic acid (ranging accessed on June 9, 2023) with autoscaling. Heatmap is used
from 0 to 493.75 μg/mL; y = 0.0012x + 0.0558, R2 = 0.997). to visualize numerical values of phenolics and glucosinolates.
For the TFC analysis, 0.5 mL of diluted extracts was mixed
with 2.0 mL of distilled water. Thereafter, 0.15 mL of 5% (v/v) 3. RESULTS
NaNO2 was added to the mixture, followed by incubation for 5 3.1. Shoot Length, Root Length, and Fresh Weight of
min at 25 °C. A 0.15 mL portion of 10% (v/v) AlCl3 was Pakchoi Plantlets. On the 24th day after sowing, shoot
added and then maintained for 15 min at room temperature. length, root length, and fresh weight were measured from four
The absorbances of each sample were measured at 415 nm. different LED light-exposed pakchoi plantlets (Figure 1). The
The flavonoid contents were quantified by using the equivalent
calibration curve of quercetin (ranging from 0 to 1000 μg/mL;
y = 0.0022x + 0.0371, R2 = 0.998). All of the results are
presented as the average of three replications and expressed in
terms of milligrams of gallic acid or quercetin equivalent per
gram of dry weight (mg gallic acid or quercetin equivalent
(GAE or QE)/g DW).
2.6. Antioxidant Assays. 2.6.1. DPPH Free Radical
Scavenging Assay. The DPPH free radical scavenging activity
was assessed as previously described.24 100 μL of each sample
extract with concentrations ranging from 31.25 to 1000 μg/mL
was placed in 96-well plates, and then, 100 μL of a 0.2 mM
DPPH solution was added. The mixtures were left in the dark
for 30 min and then measured at 517 nm using the UV−vis
spectrophotometer. The DPPH radical scavenging activity was
calculated with the following formula: DPPH radical
scavenging activity (%) = [1 − {(A0 − A1)/A2}] × 100,
where A0 is the sample absorbance, A1 is the blank absorbance,
and A2 is the control absorbance.
2.6.2. ABTS Radical Scavenging Assay. The ABTS radical
scavenging activity was assessed as previously described.25 A 7
mM concentration of ABTS powder was dissolved in a 2.5 mM
potassium persulfate solution and then incubated for 16 h in
the dark. The absorbance of the ABTS buffer solution was
adjusted to 0.7 ± 0.002 at 734 nm using distilled water. 50 μL
Figure 1. (A, B) Effect of different LED lights on shoot length and
of each sample extract with concentrations ranging from 31.25 root length in pakchoi baby leaves was determined 24 DAS in growth
to 1000 μg/mL was placed in 96-well plates, and then, 150 μL chambers. Different letters denote a significant difference in means (p
of the ABTS buffer solution was added. The mixture was < 0.05) using the Tukey test.
incubated in the dark for 10 min and then measured at 517 nm
using a UV−vis spectrophotometer. The ABTS radical
scavenging activity was calculated with the following formula: results showed that there was no statistically significant effect
ABTS radical scavenging activity (%) = {A0 − (sample − on the shoot length. However, the root length of the pakchoi
blank)}/A0 × 100, where A0 is the absorbance of ABTS mixed plantlet showed a significant difference among the different
with methanol. types of LED exposures, with the longest length in the red +
2.6.3. Reducing Power Assay. The reducing power assay blue LED light (23.80 ± 1.05 cm) and the shortest length in
was performed as previously described.26 300 μL of each the blue LED light (16.90 ± 1.15 cm). Like the shoot length,
sample extract with concentrations ranging from 31.25 to 1000 the fresh weight also showed a statistically significant difference
μg/mL was mixed with 300 μL of 0.2 M phosphate buffer, among the different LED-exposed plantlets (Figure 2). From
followed by 300 μL of 1% (v/v) C6N6FeK3 solution. After these, it can be seen that the exposure of red pakchoi baby leaf
incubation for 20 min at 50 °C, 300 μL of 10% (v/v) to different LED lights did not show any significant difference
C2HCl3O2 solution was added and then vortexed. The in shoot length, whereas the root length and fresh weight
mixtures were centrifuged at 10,000 rpm for 10 min, and showed a significant difference among the different LED
500 μL of supernatants was mixed with 500 μL of distilled treatments. However, the highest shoot length (11.10 ± 0.31
water, followed by the addition of 100 μL of 0.1% (v/v) FeCl3 cm), root length (23.80 ± 1.05 cm), and fresh weight (5.09 ±
solution. The solution was measured by using a UV−vis 0.29 g) were observed in the red + blue treatment.
spectrophotometer at 700 nm. An increase in absorption value 3.2. Glucosinolate Accumulation in Red Pakchoi Baby
at this wavelength indicates a high reducing power. Leaves. Regarding the glucosinolate content, a total of seven
2.7. Statistical Analysis. The shoot and root length, fresh glucosinolates, composed of four aliphatic glucosinolates
weight, anthocyanin, flavonoid, phenolics, antioxidant activ- (glucobrassicanapin, gluconapin, glucoberteroin, and progoi-
ities, and HPLC data were analyzed using Tukey’s multiple trin) and three indolic glucosinolates (4-methoxyglucobrassi-
range test using R at p < 0.05.27 For metabolic profiling, PCA cin, glucobrassicin, and neoglucobrassicin), were identified. In
(principal component analysis), PLS-DA (partial least-squares general, the concentrations of total glucosinolates in red
discriminant analysis), heatmap, Pearson correlation analysis, pakchoi baby leaves grown under blue and white LED lights
23422 https://doi.org/10.1021/acsomega.3c10261
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Figure 2. Effect of different LED lights on fresh weight in pakchoi
baby leaves was determined 24 DAS in growth chambers. Different
letters denote a significant difference in means (p < 0.05) using the
Tukey test.
presented no statistically significant difference (2262 ± 148
and 2228 ± 64 μg/g dry wt, respectively). However, the plants
grown under red and red + blue LED lights showed the highest
glucosinolate concentrations (2688 ± 148 and 2938 ± 217 μg/
g dry wt, respectively). Regarding the individual glucosinolate
concentrations, high concentrations of progoitrin, followed by
gluconapin and glucobrassicanapin, were found in pakchoi
plants grown under red + blue LED light, whereas in red LED
light, the highest contents were found for glucobrassicin and 4-
methoxyglucobrassicin. Moreover, higher levels of neogluco-
brassicin were found in plants exposed to red and red + blue
LED lights (Table 1).
3.3. Phenolic Compounds in Red Pakchoi Baby
Leaves. Twelve individual phenolic compounds, comprising
five hydroxycinnamic acids (caffeic acid, ferulic acid, p-
coumaric acid, chlorogenic acid, and trans-cinnamic acid),
two hydroxybenzoic acids (gallic acid and 4-hydroxybenzoic
acid), three flavonols (quercetin, rutin, and kaempferol), and
two flavan-3-ols (catechin hydrate and epicatechin), were
identified. The highest concentration of phenolic compounds
was acquired in plants exposed to red + blue LED light (1589
± 24 μg/g dry wt); however, no statistically significant
difference was observed with plants exposed to blue and white
LED lights. The lowest concentrations were obtained in plants
exposed to red LED light (1340 ± 11 μg/g dry wt). Regarding
each phenolic compound, gallic acid, catechin hydrate, p-
coumaric acid, and trans-cinnamic acid were higher in plants
exposed to red LED light. For plants grown under red + blue
LED light, the following compounds had higher concen-
trations: chlorogenic acid, epicatechin, ferulic acid, and
Table 1. Glucosinolate Concentrations (μg/g Dry wt) in Red Pakchoi Baby Leaves Grown under Different LED Lightsa
glucosinolates red
progoitrin 588.46 ± 38.46a
gluconapin 812.30 ± 64.91ab
glucobrassicanapin 487.88 ± 53.04b
glucobrassicin 247.27 ± 8.14a
4-methoxyglucobrassicin 67.10 ± 3.01a
glucoberteroin 374.48 ± 35.87a
neoglucobrassicin 110.58 ± 10.69a
total 2688.06 ± 147.82a
a
Using the Tukey test, different letters denote a significant difference in means (p < 0.05).
Article
kaempferol, whereas in the plants exposed to blue LED light,
caffeic acid, rutin, and quercetin were the highest. In the white
LED light, only one compound (4-hydroxybenzoic acid)
showed the highest accumulation (Table 2). From this result,
it can be seen that baby leaves exposed to blue, white, and red
+ blue LED do not show any significant difference in the
phenolic content; however, the highest phenolic content was
observed in the red + blue LED light-exposed leaves.
3.4. Total Anthocyanin, Flavonoid, and Phenolic
Compounds. The highest total anthocyanin content was
detected under the red + blue LED light treatment (1727 ± 29
μg of CYE/g dry wt), and the lower content was detected
under blue LED light (708 ± 97 μg of CYE/g dry wt). There
was a statistical difference between all LED light treatments
(Table 3). The white, red, and blue light treatments showed a
lower anthocyanin content compared with the red + blue LED
light treatment. The highest total flavonoid content was
detected under the red + blue LED light treatment (4920 ±
140 μg of QE/g dry wt), and the lowest content was detected
under red LED light (2910 ± 90 μg of QE/g dry wt.). There
was a statistical difference between LED light treatments
(Table 3). Compared to blue and white LED light treatment,
the total flavonoid concentration is higher in red + blue light,
but the concentration is lower in red LED light treatment.
Likewise, the highest total phenolic content was detected
under the red + blue LED light treatment (5900 ± 90 μg of
GAE/g of dry wt), and the lowest content was detected under
white LED light (3480 ± 190 μg of GAE/g of dry wt). These
findings showed that red + blue LED light exposure
significantly accumulates the total anthocyanin, flavonoid,
and phenolic content in the red pakchoi baby leaves.
3.5. Metabolic Profiling of Identified Compounds.
Seven individual glucosinolates, 12 phenolic compounds, and
total anthocyanin were identified and quantified from the
pakchoi baby leaves exposed to different carbon sources. The
heatmap was mainly grouped into two clusters, namely,
clusters 1 and 2. Cluster 1 was further subdivided into two
subgroups, 1a and 1b, and cluster 2 was also subdivided into
clusters 2a and 2b (Figure 3). Cluster 1 consisted of the
compounds highly present in the red + blue, white, and blue
LED treatments, whereas cluster 2 was separated based on the
compounds highly present in the red and red + blue LED light
treatments. Cluster 1a formed a separation based on the
highest amounts of phenolic compounds (ferulic acid,
quercetin, caffeic acid, and rutin) present in the red + blue
and blue LED light treatments, whereas cluster 1b was grouped
based on the phenolic compounds (kaempferol, 4-hydrox-
ybenzoic acid, epicatechin, and total phenolics) and total
LED light (μg/g dry wt)
blue white red + blue
469.37 ± 45.90b 472.22 ± 39.35b 661.88 ± 58.24a
771.63 ± 44.29ab 738.83 ± 43.37b 890.62 ± 110.41a
409.92 ± 53.54c 373.89 ± 21.14c 591.53 ± 25.54a
196.95 ± 7.30b 201.56 ± 13.38b 235.01 ± 17.37a
41.2 ± 3.88b 36.50 ± 6.15b 39.88 ± 0.87b
269.49 ± 19.79b 296.74 ± 14.05b 410.01 ± 26.21a
103.29 ± 5.82a 104.12 ± 3.06a 108.89 ± 7.29a
2261.90 ± 147.68b 2227.86 ± 63.88b 2937.81 ± 216.65a
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Table 2. Concentration of Phenolic Compounds (μg/g Dry Weight) in Red Pakchoi Baby Leaves Grown under Different LED
Lightsa
LED light (μg/g dry wt)
phenolic compounds red blue white red + blue
gallic acid 19.65 ± 0.98a 18.13 ± 1.45a 11.78 ± 0.25c 14.63 ± 1.38b
4-hydroxybenzoic acid 185.45 ± 0.41c 199.83 ± 17.71bc 234.19 ± 1.99a 217.89 ± 10.07ab
catechin hydrate 35.22 ± 0.65c 34.71 ± 0.98a 34.93 ± 0.78a 33.55 ± 1.36a
chlorogenic acid 54.83 ± 1.91a 52.43 ± 5.54a 53.44 ± 2.39a 56.13 ± 6.35a
caffeic acid 95.00 ± 2.35c 143.45 ± 4.90a 112.23 ± 1.49b 118.05 ± 7.08b
epicatechin 354.55 ± 6.18b 507.03 ± 17.03a 510.03 ± 17.12a 520.56 ± 30.02a
p-coumaric acid 115.35 ± 4.75a 89.79 ± 2.11c 103.81 ± 3.26ab 95.05 ± 10.61bc
ferulic acid 125.77 ± 0.27c 143.04 ± 3.48b 122.25 ± 1.18c 160.23 ± 3.19a
rutin 100.65 ± 9.50b 131.29 ± 4.81a 98.83 ± 6.62b 111.84 ± 5.20b
trans-cinnamic acid 14.67 ± 0.77a 9.13 ± 0.37b 5.21 ± 0.43c 5.63 ± 0.66c
quercetin 131.36 ± 9.47ab 141.83 ± 2.80a 123.87 ± 11.33b 131.87 ± 6.17ab
kaempferol 107.72 ± 2.06ab 103.75 ± 3.25b 121.44 ± 8.25a 123.76 ± 15.37a
total 1340.23 ± 10.92b 1574.40 ± 53.18a 1532.01 ± 32.15a 1589.19 ± 23.51a
a
Using the Tukey test, different letters denote a significant difference in means (p < 0.05).

Table 3. Total Flavonoid, Phenolic, and Anthocyanin Contents in Red Pakchoi Baby Leaves Grown under Different LEDLa
LED light type total flavonoid (μg GAE/g DW) total phenolic (μg QE/g DW) total anthocyanin (μg/g DW)
red 2905.39 ± 85.05c 4447.45 ± 93.16b 1016.41 ± 76.18c
blue 3957.80 ± 0.00b 4716.39 ± 581.80b 708.03 ± 96.62d
white 3687.18 ± 147.31b 3479.25 ± 186.33c 1225.70 ± 27.34b
red + blue 4920.00 ± 137.79a 5899.72 ± 93.16a 1726.67 ± 29.12a
a
Using the Tukey test, different letters denote a significant difference in means (p < 0.05).

According to the principal component analysis (PCA), PC1

Figure 3. Heatmap with the Euclidean distance measure of relative
contents of metabolites in pakchoi baby leaves grown under different
LED lights. The relative metabolite concentrations are represented by
the color scale (2 to −2) on the right, with high and low
concentrations denoted by red and blue, respectively.
anthocyanin compounds rich in red + blue and white
treatments. Cluster 2a consisted only of glucosinolate
compounds (gluconapin, progoitrin, glucobrassicanapin, glu-
coberteroin, and total glucosinolates), and they were
abundantly present in the red and red + blue LED light
treatments, whereas 2b was grouped based on the compounds
rich in red treatments, which consisted of both phenolic (gallic
acid, trans-cinnamic acid, and p-coumaric acid) and glucosi-
nolate (4-methoxyglucobrassicin, neoglucobrassicin, and glu-
cobrassicin) compounds. Considering these results, the
different LEDs enhanced the accumulation of different
metabolites in the red baby pakchoi leaves.
23424
and PC2 showed 33.5 and 28.5% variance, respectively (Figure
4A). In addition, partial least-squares discriminant analysis
(PLS-DA) was carried out to exploit the separation between
the LED-treated platelets. This clear separation might be due
to the gallic acid, trans-cinnamic acid, quercetin, rutin, and 4-
methoxyglucobrassicin, as well as the associated eigenvector
values (Figure 4B). Additionally, the most crucial metabolites
of variable importance in projection (VIP) analysis for the
prediction were gallic acid, 4-hydroxybenzoic acid, kaempferol,
rutin, and total anthocyanin, which led to a great separation
(Figure 5). This supports the heatmap results, which showed
that the different LEDs enhanced the accumulation of different
metabolites in red baby pakchoi leaves.
3.6. In Vitro Antioxidant Assays. The samples showed a
concentration-dependent increasing pattern. In particular,
mixed (red + blue) LED light has the highest DPPH radical
scavenging activity (45.31 ± 0.50%) at 1000 μL/mL
concentrations, followed by blue LED (40.89 ± 4.65%) and
white LED (36.74 ± 2.13%) treatments (Figure 6A). On the
other hand, red LED treatment has the lowest radical
scavenging activity (33.28 ± 1.65%) at 1000 μL/mL
concentrations. Similarly, the baby leaves revealed concen-
tration-dependent scavenging of ABTS (Figure 6B). The
mixed LED-treated baby leaf extracts at 125, 250, 500, and
1000 μL/mL concentrations showed the highest ABTS
scavenging activity compared with those of extracts under
the other LED light conditions. However, the red LED-treated
baby leaf extracts at the same concentrations showed the
lowest scavenging activity. Similarly, the reducing power assay
showed that the baby leaves under the mixed LED treatment
had the best antioxidant ability (Table 4).
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Figure 4. PCA (A) and PLS-DA (B) scores and loading plots of the metabolites found in pakchoi leaves grown under different LED lights.
Figure 5. Main components separating pakchoi leaves grown under
different LED lights are based on the VIP scores attained via the PLS-
DA model. The VIP scores are shown by the black dots, reflecting the
degree of importance of the metabolites.
4. DISCUSSION
Light is very important for plant growth and morphology.10,28
Plant growth and physiological characteristics are greatly
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influenced by light effects.29,30 In the current study, in terms of
shoot length and fresh weight, the exposure of plants to
different LED lights did not affect these variables; however,
regarding root length, plants exposed to red + blue LED light
obtained the highest values (23.80 ± 1.05 cm). These results
are consistent with a previous study on canola (B. napus subsp.
napus), which showed that the highest root length was
achieved in red + blue LED light compared to other light
treatments; this demonstrates that LED light is a valuable
source for crop development.31 However, different results with
the same crop were found; for instance, it has been reported
that the highest shoot length and fresh weight were obtained in
canola sprouts exposed to red LED light, and for root length,
no difference was found between the different LED lights.6
However, after a 60-day cultivation period of sarcandrae
(Sarcandra glabra) seedlings, the fresh weight was significantly
higher in the white and blue LED light treatments than in the
red LED light treatment. Moreover, treatments under blue
LED light resulted in considerably longer roots than under red
and white LED lights.32 Buckwheat (Fagopyrum tataricum)
sprouts were exposed to different LED lights for 10 days. The
highest lengths and fresh weights were achieved under red
LED light, whereas the lowest values were obtained after blue
LED light exposure.33 When grown under blue LED light,
cowpea (Vigna unguiculata) sprouts had 1.40 and 1.17 times
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and blue LED lights produced noticeably high glucosinolate

Figure 6. (A,B) DPPH and ABTS free radical scavenging activity in
red pakchoi baby leaves grown under different LED lights. Different
letters in the values denote statistically significant differences among
the means, using the Tukey test (p < 0.05), while values are shown as
mean ± SD.
higher shoot length and fresh weight, respectively, than those
grown under white LED light.17 Therefore, these results
showed that different LED lights have different effects on
different Brassica species. In the present study, each plant had a
different response to different types of LED light irradiation. In
pakchoi (B. rapa subsp. chinensis), exposure to different types
of LED light had no significant effect on shoot length;
however, red + blue LED light irradiation affected the root
length and fresh weight.
In the current study, PCA analysis showed that the red +
blue LED group was isolated from the other groups, and it was
due to the higher levels of glucosinolates and phenolics. Thus,
the combination of red and blue LEDs enhanced the
production of these secondary metabolites.18,19,34 In our
study, the highest glucosinolate levels were found in the leaves
exposed to red and red + blue LED lights (2937.81 ± 216.65
μg/g dw). Moreover, pretreatment of two Melissa officinalis
genotype seedlings with different LED lights showed that the
red + blue LED light enhances the accumulation of total
phenolic and anthocyanin content.35 It was observed that red
content in B. rapa subsp. pekinensis and Brassica oleracea var.
acephala.36 In addition, they found that glucobrassicanapin and
sinigrin were the predominant glucosinolates in B. rapa subsp.
pekinensis and B. oleracea var. acephala, and also that, compared
to dark light, different light conditions increased the amounts
of glucosinolates in broccoli sprouts.37 However, this indicated
that there was no statistically significant difference in the total
glucosinolate content of B. napus sprouts cultivated under
white, blue, and red LED lights. 6 The glucosinolate
concentration in B. oleracea var. acephala did not differ
significantly between the treatments using red + blue LEDs
and white LED light.38 Similar results were obtained, with no
significant difference in the total amount of glucosinolates in
the B. rapa subsp. chinensis var. parachinensis sprout grown
under white and red + blue LED light; moreover, the amount
of glucosinolates gradually decreased with increasing incuba-
tion time.5 Moreover, in this study, no significant difference in
the total glucosinolate accumulation was found in red pakchoi
baby leaves exposed to blue, white, and red + blue lights. A
similar result was found in kohlrabi sprouts exposed to
different LED lights, where no significant difference was
observed among the different LED exposures.12 According to
previous studies, plant species, a combination of light sources,
and the light intensity and spectrum may influence
glucosinolate accumulation. In addition, several studies have
reported that different Brassica cultivars and species accumu-
late different levels and profiles of glucosinolates and their
hydrolysis products based on their genotypes.39−41
In the present study, 12 phenolic compounds were identified
and measured, and the highest concentration was achieved in
plantlets grown under red + blue LED light (1589.19 ± 23.51
μg/g of dw). Furthermore, the highest individual phenolic
compound was epicatechin (520.56 ± 30.02 μg/g of dw),
which accumulated under red + blue LED light, followed by 4-
hydroxybenzoic acid under white (510.03 ± 17.12 μg/g of dw)
and blue (507.03 ± 17.03 μg/g of dw) LED lights. Our results
differ from those of Kim et al., who found that in B. rapa subsp.
pekinensis, p-hydroxybenzoic acid was the most abundant
phenolic compound quantified under blue LED light, followed
by chlorogenic acid under white LED light.42 The highest
phenolic compound level content of catechin was observed in
cowpea sprouts under blue LED light.17 However, cowpea
sprouts grown under white LED light presented the lowest
level of phenolic compounds, whereas other LED lights did not
affect the content of other phenolic compounds, such as 4-
hydroxybenzoic acid and chlorogenic acid.17 In contrast, the
highest rutin content was obtained in buckwheat sprouts
grown under blue LED light, whereas no significant difference
was observed in the accumulation of chlorogenic acid between
sprouts grown under other LED light treatments.33 In

Table 4. Reducing Power Assay of Red Pakchoi Baby Leaves Grown under Different LED Lightsa
absorbance in 700 nm
31.25 62.5 125 250 500 1000
ascorbic acid 0.28 ± 0.00a 0.49 ± 0.01a 0.91 ± 0.02a 1.67 ± 0.02a 2.28 ± 0.02a 2.31 ± 0.00a
red 0.08 ± 0.00c 0.09 ± 0.00b 0.09 ± 0.00c 0.11 ± 0.00c 0.13 ± 0.00c 0.19 ± 0.00e
blue 0.09 ± 0.00b 0.09 ± 0.00b 0.09 ± 0.00bc 0.11 ± 0.00c 0.14 ± 0.00c 0.22 ± 0.00d
white 0.09 ± 0.00bc 0.09 ± 0.00b 0.11 ± 0.00bc 0.14 ± 0.00b 0.20 ± 0.00b 0.32 ± 0.00c
red + blue 0.08 ± 0.00bc 0.09 ± 0.00b 0.11 ± 0.00b 0.15 ± 0.00b 0.22 ± 0.00b 0.37 ± 0.00b

a
Using the Tukey test, different letters denote a significant difference in means (p < 0.05).

23426 https://doi.org/10.1021/acsomega.3c10261
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Agastache rugosa seedlings, white LED light has been
demonstrated to promote the accumulation of phenolics.43
Moreover, red, blue, or red + blue LED light had no impact on
phenolic compound accumulation in tartary buckwheat
sprouts.44 Additionally, fluorescent light exposure resulted in
higher concentrations of ginsenoside-Rb1 and ginsenoside-
Rg1.45 In addition, Aronia melanocarpa, Aronia punifolia, and
Aronia arbutifolia under blue LED light increased phenolic acid
production in their shoots.46 Similarly, chlorogenic acid was
found in the callus of Peucedanum japonicum,47 and a high total
phenolic content was found in the callus of Ocimum
bassilicum.48 Blue light treatment resulted in the highest
accumulation of phenolic compounds in kohlrabi sprouts.12
However, in this study, red + blue light exposure showed the
highest phenolic accumulation. These results show that
different LED lights have different effects on phenolic
compounds, based on the plant genus.
Anthocyanin can change both quantitatively and qualita-
tively depending on the lighting conditions.49 In the present
study, we found color changes in leaves exposed to red + blue
LED light (1726.67 ± 29.12 μg/g dw), which in turn had the
highest anthocyanin concentration. This result is in line with
studies that observed that almost 90% of the purple, blue,
orange, and red light was absorbed by plants.50 In a study using
three different types of LED lights (white, blue, and red), it
was found that the total anthocyanin content in B. juncea
seedlings varied according to the duration of LED
irradiation.51 The study found that the seedlings had the
highest total anthocyanin content after 3 weeks of treatment
with blue LED light, followed by red LED light treatment. In
contrast, blue LED light enhances the accumulation of
anthocyanin compared with white, red, or red + blue LED
light in Fagopyrum tataricum sprouts.33 In this study, the total
flavonoid and phenolic contents varied depending on the LED
conditions. In particular, the highest levels of the total
flavonoid (4920.00 ± 137.79 μg GAE/g dw) and phenolic
(5899.72 ± 93.16 μg QE/g dw) contents were obtained in the
baby leaves under mixed (red + blue) LED light. These
findings were consistent with those from previous studies.
Jarerat et al. conducted the measurement of TPC and TFC
contents accumulated in the peel and flesh of eggplants
exposed to various types of LEDs (red, blue, and red + blue),
indicating that mixed LED (red + blue) was found to be
effective in the accumulation of total phenolics and total
flavonoids in the flesh and peel of eggplants.52 Furthermore,
Gam et al. reported that mixed (blue + red) LEDs accumulated
the highest total flavone content in Anoectochilus roxburghii
compared to other light conditions (fluorescent lamp, blue-red,
blue:red:white ratios of 1:5:1 and 1:4:2).53
In this study, red pakchoi baby leaves exposed to mixed LED
(red + blue) had the highest levels of most phenolic
compounds, followed by baby leaves under blue LED. It
might be due to the activation of the HY5 transcription factor
by blue LED lights. HY5 is known to regulate flavonoid
biosynthesis under visible light. Particularly, HY5 transcription
can be activated by blue light and activates the expression of
MYB transcription factors (MYB12 and MYB75) involved in
flavonoid biosynthesis. These MYB factors activate the
expression of flavonoid biosynthesis genes and lead to an
increase in the production of phenolic compounds.51 Hence,
mixed LED (red + blue) and blue LED are considered optimal
lights for the production of phenolics in red pakchoi baby
leaves.
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Article
Plant antioxidant activities are influenced by light conditions.
In this study, the antioxidant ability of red pakchoi baby leaves
exposed to four different LED light conditions was determined
using three different antioxidant assays. The DPPH and ABTS
scavenging activities of red pakchoi baby leaves grown under
blue and mixed LEDs were relatively higher than those of baby
leaves grown under white and red LEDs. The higher
antioxidant activities were due to the higher levels of phenolics,
anthocyanins, flavonoids, and glucosinolates. The findings were
consistent with a previous study reporting that blue LED light
increased the accumulation of anthocyanins and phenolic acids
in Brassica juncea sprouts, and their extracts showed a higher
antioxidant capacity.51 In addition, Jarerat et al. reported that
the peel of eggplant exposed to red + blue LED lights had the
highest DPPH inhibition and ferric-reducing antioxidant
power.52 As a result, for the accumulation of secondary
metabolites (glucosinolates, flavonoids, and phenolic com-
pounds) and antioxidant activity, plants exposed to a
combination of red + blue LED light proved to be the most
effective.
5. CONCLUSIONS
In the present study, each plant had a different response to
different types of LED light irradiations. In pakchoi, exposure
to different types of LED lights had no significant effect on
shoot length; however, red + blue LED light irradiation
affected the root length and fresh weight. Regarding the
concentrations of glucosinolates, flavonoids, phenolics, antho-
cyanin, and antioxidant activity, the current results indicate
that plants exposed to a combination of red + blue LED light
proved to be the most effective artificial light source.
Therefore, this study might establish an effective strategy for
improving the antioxidants and the content of some
phytochemicals, namely, glucosinolates, flavonoids, phenolic,
and anthocyanin production. The metabolic profiling results
will provide valuable insights into helping us determine
whether pakchoi baby leaves exposed to different LED lights
contain high levels of phenolics, glucosinolates, anthocyanin,
and antioxidants. This information holds significance for
assessing their suitability for human consumption. Further,
this study suggested that polyphenols and glucosinolates in
pakchoi baby leaves under different LED light sources can
work synergistically as antioxidants. Further analysis of
metabolite relationships may provide an insight into pakchoi’s
secondary metabolite biosynthesis pathway under different
LED illuminations.
■ ASSOCIATED CONTENT
Data Availability Statement
The data underlying this study are available in the published
article and its Supporting Information.
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.3c10261.
Additional experimental materials and methods; Table
S1, retention times of glucosinolates; Tables S2,
calibration curve of phenolic compounds (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Yong Suk Chung − Department of Plant Resources and
Environment, Jeju National University, Jeju 63243, Republic
https://doi.org/10.1021/acsomega.3c10261
ACS Omega 2024, 9, 23420−23430
ACS Omega http://pubs.acs.org/journal/acsodf
of Korea; Email: yschung@jejunu.ac.kr; Fax: +82-64-755-
6130
Hyeon Ji Yeo − Department of Crop Science, Chungnam
National University, Daejeon 34134, Republic of Korea;
orcid.org/0000-0002-5474-8204; Email: guswl7627@
gmail.com
Authors
Leonel Tarcisio da Cristina Bungala − Department of Crop
Science, Chungnam National University, Daejeon 34134,
Republic of Korea; Mozambique Agricultural Research
Institute, Chimoio 0606-01, Mozambique
Sang Un Park − Department of Crop Science, Chungnam
National University, Daejeon 34134, Republic of Korea;
Department of Smart Agriculture Systems, Chungnam
National University, Daejeon 34134, Republic of Korea;
orcid.org/0000-0003-2157-2246
Bao Van Nguyen − Department of Smart Agriculture Systems,
Chungnam National University, Daejeon 34134, Republic of
Korea
Jinsu Lim − Department of Bio-AI Convergence, Chungnam
National University, Daejeon 34134, Republic of Korea
Kihyun Kim − Department of Crop Science, Chungnam
National University, Daejeon 34134, Republic of Korea
Jae Kwang Kim − Division of Life Sciences, College of Life
Sciences and Bioengineering, Incheon National University,
Incheon 22012, Republic of Korea; Convergence Research
Center for Insect Vectors, Incheon National University,
Incheon 22012, Republic of Korea; orcid.org/0000-0003-
2692-5370
Chang Ha Park − Department of Biological Sciences,
Keimyung University, Daegu 42601, Republic of Korea
Anh Tuan Le − Department of Plant Resources and
Environment, Jeju National University, Jeju 63243, Republic
of Korea; orcid.org/0000-0002-9584-7807
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.3c10261
Author Contributions
◆ Photosynthetica 2017, 55, 85−95.
L.T.d.C.B. and S.U.P. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education
(2019R1A6A1A11052070). L.T.d.C.B. would like to express
his deepest gratitude to the National Institute for International
Education (NIIED) through the Global Korea Scholarship
(GKS-G-2020-593) for providing funding for his studies.
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https://doi.org/10.1021/acsomega.3c10261
ACS Omega 2024, 9, 23420−23430
ACS Omega http://pubs.acs.org/journal/acsodf Article

LED lights promote growth and flavonoid accumulation of

Anoectochilus roxburghii and are linked to the enhanced expression
of several related genes. Plants 2020, 9 (10), 1344.

23430 https://doi.org/10.1021/acsomega.3c10261
ACS Omega 2024, 9, 23420−23430